AMP-activated protein kinase (AMPK) is the "fuel gauge" of mammalian cells and serves as a major regulator of glucose and lipid metabolism in response to metabolic stress. AMPK is implicated in type 2 diabetes, obesity, and cardiac disease, and it is a target of the hormones leptin and adiponectin and the antidiabetic drug metformin. The upstream kinases that activate AMPK, the AMPKKs, remained unknown until recently. Our studies of the upstream kinases of the yeast (Saccharomyces cerevisiae) ortholog of AMPK, Snf1 protein kinase, led us to identify the LKB1 tumor suppressor kinase as the major AMPKK in liver. This proposal explores an innovative use of the yeast genetic system to identify other human AMPKKs. In yeast mutant cells lacking the native upstream kinases for Snf1, expression of LKB1 activates Snf1. New genetic assays will be developed to take advantage of this functional conservation between the AMPK and Snf1 pathways in order to readily detect and select for AMPKK function in yeast cells. The key criterion for utility of the assay will be ability to recover cDNA clones expressing LKB1 from a liver cDNA expression library. Human cDNA expression libraries from tissues in which AMPK plays important roles will then be used to select for kinases that activate Snf1 in this yeast genetic assay. To validate candidate kinases as potential AMPKKs, their ability to phosphorylate and activate AMPK will be verified. AMPKKs may be specific to particular tissues and may respond to specific hormones or signals generated by particular metabolic stress conditions. Hence, AMPKKs are potentially more specific therapeutic targets for treatment of diabetes and obesity than AMPK. [unreadable] [unreadable]